Significant biological interest has surrounded the transcription factor NF-vJ3 for the following reasons: (i) its inducibility by cytokines and other immunological/inflammatory mediators, (ii) its interesting regulation through interactions with the Ivd3 inhibitory proteins, and (iii) its critical involvement with a range of diseases (both inflammatory and oncological, Baldwin, J. Clin. Inv. 107, 3-6). Studies on NF-vJ3 regulation have led to a fairly cohesive model which involves: the activation of the Ild3 kinase (IKK, containing IKKcc, IKK[3, IKKy), phosphorylation of Ivd3 by IKK, Ivd3 degradation, and nuclear accumulation of NF-vJ3. In this regard, one of the IKK subunits (IKK[3) is required for TNF induction of NF-vJ3 through its ability to control Ivd3 phosphorylation. The lack of involvement of IKKcz in this pathway has led to the proposal that this IKK subunit is not involved in the cytokine- controlled NF-vd3 regulatory pathway. However, emerging data has revealed a more complex and significantly more interesting view of NF-vJ3 regulation and IKK function. Thus, it has been shown that IKK(z is required for the induction of NF-vJ3-dependent gene expression downstream of TNF-induced signaling and that GSK-3[3 (the kinase involved in Wnt signaling) controls NF-vJ3 functional activity downstream of IKB degradation and NF-vJ3 nuclear accumulation. These results indicate that regulation of NF-vd3 transcriptional competence is controlled at the nuclear level in addition to mechanisms which regulate release from Ivd3. Additionally, IKK(z (but IKK[_) is required for TLI3R- induced signaling controlling NF-vd32 activation and the control of specific chemokine/cytokine gene expression. Further evidence for functional differences between IKKcz and IKK[3 is provided by the phenotype of the IKKc_ null animal which exhibits skin and skeletal abnormalities associated with a block on keratinocyte differentiation that may be independent of NF-vd3. Our preliminary data demonstrate new findings which are likely to explain major mechanisms associated with the regulation of NF-vd3-dependent, and potentially NF-vJ3-independent, gene expression. Thus, we have found that IKKcz is induced to accumulate in the nucleus following cytokine treatment of a variety of cells. Surprisingly, we find that IKK_ and IKKI3 are both induced to associate with the promoter regions of the NF4d3-regulated genes. Evidence is presented that IKK_ controls cytokine inducible histone H3 serl0 phosphorylation, an initiating event in the control of histone modification described by the 'histone code hypothesis.'Furthermore, we find that IKKcz is required to control epidermal growth factor (EGF)-induced histone H3 serl0 phosphorylation and that GSK-313 controls the phosphorylation of the p65 subunit on ser536. We propose to analyze the structural and regulatory features of IKKcz and IKK[3 involved in modulating chromatin function extending the hypothesis to the existence of separate IKKo_ and IKK[3-containing complexes regulating chromatin modification. We also hypothesize that IKK(z is involved in the control of chromatin modification for genes that are not NF-vd3-dependent, potentially explaining the requirement of IKKcz in controlling keratinocyte differentation. Finally, we propose to determine the role of GSK-3[3 in controlling p65 ser56 phosphorylation, relating this to the potential involvement of the IKKs. Additionally, using restoration experiments in p65 null cells and knock-in technology we propose to analyze the requirement of p65 ser536 phosphorylation in overall NF-v-J3 function. These experiments have the potential to provide new, significant insight into nuclear mechanisms involved in controlling NF-vJ3-dependent (and possibly independent) gene expression through control of chromatin modification and assembly of active transcription complexes.